Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T‐cell lymphoma

Abstract Leukemic cutaneous T‐cell lymphomas (L‐CTCL) are lymphoproliferative disorders of skin‐homing mature T‐cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L‐CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L‐CTCL, which correlated with the increased clonal T‐cell count in the blood. Dual inhibition of STAT3/5 using small‐molecule degraders and multi‐kinase blockers abolished L‐CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L‐CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti‐leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L‐CTCL.

A-D Immunoblot showing total and pY-STAT3/pY-STAT5 levels (i.e., phospho-Tyr (705)-STAT3 and phospho-Tyr (694/699)-STAT5A/B) in (A) Myla cells upon 6 h treatment with JPX-0750 and (B) Hut78 cells upon 24 h treatment with JPX-0750. (C) Myla and (D) Hut78 cells were treated with 24 h IQDMA. HSC70 was used as loading control. The normalized phospho-and total protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown. E RT-qPCR for STAT5 target genes in the SeAx cell line after 24 h treatment with JPX-0750. Gene expression was normalized to GAPDH. Statistical significance was calculated using two-way ANOVA with multiple comparisons. P-value: < 0.05 (*), < 0.01 (**), < 0.001 (***), and 0.0001 (****). P-value summaries are provided in Appendix Table S1. Error bars represent mean AE SD. One experiment performed in triplicates is shown. F, G Cleaved and total PARP protein levels in Myla cells treated with (F) JPX-0750 for 6 h and (G) IQDMA for 24 h. HSC70 was used as loading control. The normalized total protein levels, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown. H, I Immunoblotting for cleaved caspase 3 levels in SeAx cells after 24 h treatment with (H) JPX-0750 and (I) IQDMA. b-Actin or HSC70 were used as loading controls.
The normalized total protein levels, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.
Source data are available online for this figure.  Figure EV3. In-depth analysis of IQDMA shows its multi-kinase inhibitory function.
A Thermal shift profiles of 2 lM STAT5B with 100 lM IQDMA or 100 lM STAT5-binding peptide (positive control). The local maxima indicate the melt temperature of the protein (T m AE 0.3°C). One representative out of two independent experiments performed in technical triplicates is shown. B Fluorescence polarization assay using 180 nM STAT5B with 100 lM IQDMA or 100 lM peptide (positive control). The positive control exhibits a single-site displacement profile. Error bars represent mean AE SD. Three independent experiments were performed. C IQDMA structure. D Subcellular fractions of SeAx cells treated with IQDMA for 18 h and immunoblotted for pY-STAT3 (phospho-Tyr (705)-STAT3) and total STAT3. The cells were collected 30 min after 5 ng/ml IL-2 cytokine addition. a-Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho-and total STAT3 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One experiment was performed. E Bar graph showing the cellular pathways that are most affected by IQDMA treatment as determined by network enrichment analysis. F Subcellular fractions of SeAx cells treated with FRAx597 for 18 h and immunoblotted for pY-STAT3/pY-STAT5 levels (i.e., phospho-Tyr (705)-STAT3 and phospho-Tyr (694/699)-STAT5A/B) and total STAT3/5 levels. The cells were collected 30 min after 5 ng/ml IL-2 cytokine addition. a-Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho-and total STAT3/5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One experiment was performed.
Source data are available online for this figure. Heatmap showing IC 50 values upon treatment of primary PBMCs isolated from healthy controls with JPX-0750, IQDMA, and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48 h drug treatment is shown. The relative STAT3/5 log 2 ratio value is depicted in gray.
Source data are available online for this figure.
▸ Figure EV5. The PAK kinase inhibitor inhibits malignant cell dissemination into the lymph nodes in vivo.
A-C Flow cytometric analysis of malignant cell dissemination into (A) lymph nodes, (B) liver, and (C) kidney (Vehicle: n = 3, JPX-0750: n = 3, IQDMA: n = 3; Vehicle: n = 2, FRAx597: n = 3, with n representing the number of analyzed tumors per group) as measured by the percentage of human CD45 + cells in the respective organ. Error bars represent mean AE SEM. Statistical significance was calculated using a two-tailed paired t-test with Welch's correction. P-value: < 0.05 (*). D-F H&E and IHC analyses of Hut78-derived tumors treated with JPX-0750 (n = 3), IQDMA (n = 2) or vehicle (n = 4), and FRAx597 (n = 3) or vehicle (n = 2), and stained with (D) H&E and (E) cleaved caspase 3 (CC3) to detect cell death and (F) CD31 vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100, and 25 lm. Violin plots show the perimeter of the annotated tumor cell infiltration/expansion region into the intradermal and subcutaneous region of the skin, as well as quantification of the percentage of CC3 + cells in the tissue. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using a two-tailed paired t-test with Welch's correction. P-value: < 0.05 (*), 0.0001 (****). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.
Source data are available online for this figure.